PROJECT SUMMARY In this proposal, EpiCypher is partnering with Dr. Jian Jin, a leader in medicinal chemistry and epigenetic drug discovery, to develop the first epigenetics-focused chemical library for histone methylation. Small-molecule inhibitors that target histone methylation are an emerging class of therapeutics for cancer treatment. This hot bed of drug development is fueled by the identification of numerous histone lysine methyltransferase (KMT) and demethylase (KDM) enzymes that when dysregulated play driving roles in cancer initiation/progression, processes that can be reversed upon genetic or pharmacological modulation of aberrant enzyme activity. EpiCypher is pioneering the development of nucleosome-based epigenetic reagents and assays to accelerate identification of new therapeutics that target chromatin regulation. Nucleosomes are the repeating units of chromatin comprised of a histone octamer and 147bp DNA. The use of physiological substrates is ideal for biochemical inhibitor assays as natural substrates are more likely to identify compounds with target-specific activity. For example, the KMT NSD2 (a high value drug target for multiple myeloma) is only active when targeting nucleosome substrates in vitro and in vivo. Similarly, the KDM4A subfamily of KDMs prefers nucleosome substrates methylated at H3K36. To meet this need in the market, EpiCypher is commercializing development of recombinant nucleosomes carrying specific histone post-translational modifications (PTMs) (termed designer nucleosomes or ?dNucs?) for next-generation HTS assay development. Despite our recent advances in recombinant nucleosome-based assays, therapeutic development for chromatin- targeting enzymes is currently limited by the lack of epigenetics-focused chemical libraries. Indeed, current diversity libraries available today were largely engineered to target other enzyme classes (such as kinases and GPCRs), a result of historical inquiry (by various companies/researchers) rather than targeted design. For this study, we will focus on compounds targeting histone lysine methylation regulators (KMTs and KDMs), which are excellent drug targets due to their strong association with cancer pathogenesis. We will employ a unique strategy to develop novel compounds based on existing tool inhibitor scaffolds for KMT and KDM enzymes. The resulting focused compound sets developed here will be rigorously validated using KMTs and KDMs on EpiCypher?s nucleosome-based AlphaNucTM platform. Using EpiCypher?s unique access to a broad range of highly validated reagents for epigenetic enzyme assays, compounds demonstrating inhibition against target enzymes will be tested against a panel of KMTs and KDMs to assess their specificity. This proof of concept study is focused on histone lysine methylation due to the large number of enzymes associated with disease. In Phase II, we will vastly expand our focused compound library as well as expand to target disease-relevant arginine methyltransferases.